Höcker, B.; Lechner, H.

ChemBioChem 2021, 22, 1573-1577, 10.1002/cbic.202000880

An artificial cofactor based on an organocatalyst embedded in a protein has been used to conduct the Baylis-Hillman reaction in a buffered system. As protein host, we chose streptavidin, as it can be easily crystallized and thereby supports the design process. The protein host around the cofactor was rationally designed on the basis of high-resolution crystal structures obtained after each variation of the amino acid sequence. Additionally, DFT-calculated intermediates and transition states were used to rationalize the observed activity. Finally, repeated cycles of structure determination and redesign led to a system with an up to one order of magnitude increase in activity over the bare cofactor and to the most active proteinogenic catalyst for the Baylis-Hillman reaction known today.

Pordea, A.

J. Inorg. Biochem. 2021, 220, 111446, 10.1016/j.jinorgbio.2021.111446

Artificial metalloenzymes result from the insertion of a catalytically active metal complex into a biological scaffold, generally a protein devoid of other catalytic functionalities. As such, their design requires efforts to engineer substrate binding, in addition to accommodating the artificial catalyst. Here we constructed and characterised artificial metalloenzymes using alcohol dehydrogenase as starting point, an enzyme which has both a cofactor and a substrate binding pocket. A docking approach was used to determine suitable positions for catalyst anchoring to single cysteine mutants, leading to an artificial metalloenzyme capable to reduce both natural cofactors and the hydrophobic 1-benzylnicotinamide mimic. Kinetic studies revealed that the new construct displayed a Michaelis-Menten behaviour with the native nicotinamide cofactors, which were suggested by docking to bind at a surface exposed site, different compared to their native binding position. On the other hand, the kinetic and docking data suggested that a typical enzyme behaviour was not observed with the hydrophobic 1-benzylnicotinamide mimic, with which binding events were plausible both inside and outside the protein. This work demonstrates an extended substrate scope of the artificial metalloenzymes and provides information about the binding sites of the nicotinamide substrates, which can be exploited to further engineer artificial metalloenzymes for cofactor regeneration.

Arseniyadis, S.; Campagne, J.; Smietana, M.

Chem. Eur. J. 2020, 26, 3519-3523, 10.1002/chem.202000516

While artificial cyclases hold great promise in chemical synthesis, this work presents the first example of a DNA-catalyzed inverse electron-demand hetero-Diels–Alder (IEDHDA) between dihydrofuran and various α,β-unsaturated acyl imidazoles. The resulting fused bicyclic O,O-acetals containing three contiguous stereogenic centers are obtained in high yields (up to 99 %) and excellent diastereo- (up to >99:1 dr) and enantioselectivities (up to 95 % ee) using a low catalyst loading. Most importantly, these results show that the concept of DNA-based asymmetric catalysis can be expanded to new synthetic transformations offering an efficient, sustainable, and highly selective tool for the construction of chiral building blocks.

Song, W.J.; Tezcan, F.A.

J. Am. Chem. Soc. 2017, 139, 16772-16779, 10.1021/jacs.7b08981

We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn4:C96RIDC14, which was constructed from cytochrome cb562 building blocks via a metal-templating strategy. Previously, we reported that Zn4:C96RIDC14 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn8:A104AB34, which in turn displayed catalytic activity for the hydrolysis of activated esters and β-lactam antibiotics. Zn8:A104AB34 was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn8:A104/G57AB34 which displayed enzyme-like Michaelis–Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn4:C96RIDC14 could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn8:AB54, which houses similar, unsaturated Zn coordination sites to those in Zn8:A104/G57AB34, but in completely different microenvironments. Zn8:AB54 displays Michaelis–Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn8:AB54 revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in β-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.

Yilmaz, F.

Process Biochem. 2018, 72, 71-78, 10.1016/j.procbio.2018.06.005

Carbonic anhydrase (carbonic dehydratase) (CA) is a metalloenzyme that contains zinc (Zn2+) ion in its active site. CA catalyzes the reversible conversion of carbon dioxide and water to bicarbonate and protons. Zn2+ ions, which are present in the active site of the enzyme, interact with the substrate molecules directly and cause catalytic effect. In this study, a nano-enzyme system was designed in aqueous solutions at room temperature and under nitrogen atmosphere to use the CA enzyme without any pre-treatment and deformation in its structure. The novel concept ANADOLUCA (AmiNo Acid (monomer) Decorated and Light Underpinning Conjugation Approach) was used for this process, nano CA enzyme of size 93 nm was synthesized. The activity of the synthesized nano CA was measured following the change in absorbance during the conversion of 4-nitrophenylacetate (NPA) to 4-nitrophenylate ion at 348 nm for a period of 10 min at 25 °C compared with free CA enzyme. Km and Vmax values for nano CA enzyme were found to be 0.442 mM and 1.6 × 10−3 mM min-1, respectively, whereas Km and Vmax values for free CA were found to be 0.471 mM and 1.5 × 10−3 mM min-1, respectively. In addition to these, the Zn2+ ion present in the active site of the nano CA enzyme was replaced by rodium metal. This nanorodium-substituted CA has been investigated as a new reductase enzyme for the stereoselective hydrogenation of olefins. Then, the Zn2+ ion in the active site of the nano CA enzyme was replaced with manganese metal to enhance the enzyme structure, thereby gaining characteristics of peroxidase. This newly synthesized nano manganese-substituted CA enzyme was investigated for its role as a peroxidase, which could be an alternative for hydrogen peroxidases.

Song, W.J.

Chem. Sci. 2021, 12, 5091-5101, 10.1039/d0sc06823c

Directed evolution has provided us with great opportunities and prospects in the synthesis of tailor-made proteins. It, however, often requires at least mid to high throughput screening, necessitating more effective strategies for laboratory evolution. We herein demonstrate that protein symmetry can be a versatile criterion for searching for promising hotspots for the directed evolution of de novo oligomeric enzymes. The randomization of symmetry-related residues located at the rotational axes of artificial metallo-β-lactamase yields drastic effects on catalytic activities, whereas that of non-symmetry-related, yet, proximal residues to the active site results in negligible perturbations. Structural and biochemical analysis of the positive hits indicates that seemingly trivial mutations at symmetry-related spots yield significant alterations in overall structures, metal-coordination geometry, and chemical environments of active sites. Our work implicates that numerous artificially designed and natural oligomeric proteins might have evolutionary advantages of propagating beneficial mutations using their global symmetry.